Pine needle (Pinus massoniana Lamb.) of large quantity in China and health benefit makes its application on pharmaceutical and food industry in high demand. The chemical composition of pine needle aqueous extract (PNAE) analyzed by gas chromatograph-mass spectrometry revealed that among more than 10 compounds in PNAE, organic acids were over 76.92%, with acetic acid being 25.20%, hexadecanoic acid 18.19%, and 2-methoxy-4-vinylphenol 16.44%. Ultra-performance liquid chromatography-mass spectrometry disclosed other 5 short chain organic acids, including citric acid, succinic acid, malonic acid, malic acid, and oxalic acid. The antibacterial activity of PNAE on common spoilages and pathogenic bacteria showed that the growth of Bacillus subtilis, Staphylococcus aureus, Bacillus cereus, Micrococcus luteus, Escherichia coli, and Proteus vulgaris were inhibited significantly, with minimum inhibitory concentrations and minimum bactericidal concentrations being 3.8-15 and 7.5-30 mg/mL, respectively. Our findings suggested that pine needles with effective and safe antibacterial components possess the potential to be developed into efficacious natural antiseptic products for food disinfection and medical purpose.